糖肾平通过TGF-β1-Smad2/3-ILK信号通路干预高糖＋LPS诱导足细胞上皮间质转分化的分子机制研究

也页目的：探讨糖肾平对高糖环境下脂多糖（ lipopolysaccharide,LPS）刺激足细胞上皮间质转分化的影响,并探讨其作用机制。方法：以体外培养大鼠肾小球足细胞为研究对象,以高糖（25 mmol/L）、LPS（1μg/mL）刺激足细胞建立模型,分为正常组、高糖组、高糖＋LPS组、厄贝沙坦组、抑制剂组、糖肾平小、中、大剂量组。采用Western blotting及RT-PCR方法检测足细胞中转化生长因子-β1（ transforming growth factor-β1,TGF-β1）、Smad2/3、整合素连接激酶（ integrin-linked kinase, ILK）、CD2相关蛋白（CD2AP）、α-平滑肌肌动蛋白（α-Smooth muscle actin,α-SMA）的表达水平。结果：与正常组比较,高糖组和高糖＋LPS组足细胞TGF-β1、ILK、α-SMA蛋白及其mRNA表达明显增加（P〈0.01）,P-Smad2/3蛋白及Smad2/3 mRNA表达明显增加（P〈0.01）,CD2AP蛋白及其mRNA表达明显减少（P〈0.01）;与高糖＋LPS组比较,厄贝沙坦组足细胞P-Smad2/3、α-SMA蛋白表达明显减少（P〈0.01）,TGF-β1蛋白表达减少（P〈0.05）,TGF-β1,Smad2/3,α-SMAmRNA表达明显减少（P〈0.01）,ILK mRNA表达减少（P〈0.05）,CD2AP蛋白及其mRNA表达明显增加（P〈0.01）;糖肾平大、中、小各剂量组足细胞TGF-β1蛋白表达明显减少（P〈0.01）,糖肾平大剂量组足细胞TGF-β1 mRNA表达减少（P〈0.05）,小、中剂量组表达明显减少（P〈0.01）;糖肾平小、中、大各剂量组足细胞P-Smad2/3蛋白及Smad2/3mRNA表达均明显减少（P〈0.01）;糖肾平小、大剂量组足细胞ILK mRNA表达减少（P〈0.05）,中剂量组表达明显减少（P〈0.01）;糖肾平大剂量组足细胞α-SMA蛋白及其mRNA表达减少（P〈0.05）;糖肾平小、中、大各剂量组足细胞CD2AP蛋白及其mRNA表达均明显增加（P〈0.01）。结论：糖肾平能够降低足细胞TGF-β1、ILK蛋白及mRNA表达,降低P-Smad2/3蛋白及Smad2/3 mRNA表达,升高足细胞标志物CD2AP蛋白及mRNA表达,降低间充质细胞标志物α-SMA蛋白及mRNA表达,通过抑制TGF-β1-Smad2/3-ILK信号通路的激活减少足细胞转分化,保护足细胞,可能是其防治糖尿病肾病的作用机制之一。     

血管紧张素Ⅱ灌注诱导nephrin表达改变与足细胞凋亡

目的 研究血管紧张素Ⅱ（AngⅡ）灌注对大鼠足细胞裂隙膜分子nephrin表达及足细胞凋亡的影响，以及探讨AngⅡ引起蛋白尿及肾小球硬化的机制。方法 36只雄性Sprague Dawley大鼠分为AngⅡ灌注组(400 ng&#8226;kg-1&#8226;min-1)、生理盐水灌注组和正常对照组，测定28 d内大鼠血压及尿蛋白。分别于14、28 d处死动物取肾，观察组织学改变，并用免疫荧光、免疫电镜检测nephrin分布。RT-PCR及Western印迹法分别检测nephrin mRNA及蛋白表达。TUNEL法检测足细胞凋亡。结果 (1) AngⅡ灌注组大鼠血压升高，14 d达峰值并维持该水平至28 d；AngⅡ灌注7 d即出现蛋白尿，并持续增加。(2) AngⅡ灌注14 d时，足细胞裂隙膜变窄；灌注28 d时，足突增宽及节段性融合，部分足细胞有凋亡小体形成，少数肾小球出现节段性硬化。TUNEL法检测发现足细胞凋亡[（2.7±1.6）个/肾小球切面]，凋亡数与蛋白尿量呈正相关（r = 0.86，P < 0.01）。(3) AngⅡ灌注14 d时，肾皮质nephrin mRNA及蛋白表达上调（P < 0.05）。nephrin由正常的沿毛细血管袢线状分布向粗颗粒、团块状分布模式转变。AngⅡ灌注28 d时，肾皮质nephrin mRNA及蛋白表达下降（P < 0.05），且nephrin蛋白表达与足细胞凋亡数呈负相关（r = －0.63，P < 0.01）。 结论 AngⅡ灌注诱导的nephrin表达及分布改变可能导致了足细胞凋亡及肾小球硬化的发生与发展。     

替米沙坦对老年糖尿病大鼠足细胞黏附的保护作用

目的 探讨替米沙坦对足细胞黏附的保护作用。 方法 用20月龄SD大鼠制备糖尿病模型，随机分为糖尿病组和替米沙坦治疗组，另设老年和6周龄对照组。于实验第4、8、12周末收集大鼠24 h尿，监测血压。12周后行组织病理学检查，检测尿白蛋白（24 h）和血、尿肌酐。以间接免疫荧光-hoechst 33342双染检测尿中足细胞数量。RT-PCR、免疫组化和Western印迹等方法检测肾小球中整合素α3、整合素连接激酶(ILK) mRNA和蛋白表达。 结果 随实验进程，老年糖尿病大鼠尿白蛋白、血压和尿中足细胞数量明显高于对照组(P <0.05)，且出现较为明显的病理学损害。肾小球整合素α3 mRNA及蛋白表达量减少， ILK表达明显增加(P < 0.05)。第12周，整合素α3蛋白表达与ILK表达呈负相关 (P < 0.05)。尿足细胞数量、尿白蛋白程度与ILK表达呈正相关 (P < 0.05)。替米沙坦明显减轻肾脏病理损害，增加整合素α3表达，降低ILK表达，减少尿足细胞数量。 结论 老年糖尿病大鼠足细胞的黏附能力下降，尿足细胞排泄增加。替米沙坦可增强足细胞黏附，对足细胞存活起重要作用。     

活性维生素D改变FSGS大鼠肾小球整合素连接激酶的表达

目的观察整合素连接激酶（ILK）在局灶节段性肾小球硬化（FSGS）大鼠肾小球的表达以及活性维生素D[1,25-（OH2D3]对其表达的影响。方法SD大鼠随机分为对照组、模型组和治疗组,每组10只。采用左肾摘除、阿霉素重复注射诱导FSGS大鼠模型。治疗组皮下埋置渗透性微量泵,给予1,25-（OH）：D30．03ng·g^-1·d^-1,连用8周。检测3组大鼠尿蛋白、尿足细胞。血清白蛋白（SA）及半胱氨酸蛋白酶抑制剂（CysC）,测定。肾小球硬化指数（GSI）,电镜检测每视野足细胞数、足突平均宽度,间接免疫荧光检测肾小球ILK蛋白的表达,WT-1免疫组化染色观察每个肾小球足细胞数目。结果①与对照组相比较,模型组大鼠尿蛋白、尿足细胞、Cyst、GSI明显增加,SA、肾小球足细胞数目减少,足突宽度增加,肾小球ILK表达明显降低;②与模型组相比较,治疗组尿蛋白、尿足细胞排泄明显减少,GSI明显降低,肾小球足细胞数目增多,足突宽度减小,肾小球ILK表达明显增加。结论1,25-（OH2）D3可增加照;S大鼠肾小球ILK的表达,减少足细胞脱落,维持肾小球足细胞数量。     

尿毒清对阿霉素肾病大鼠肾纤维化的保护作用

目的 研究尿毒清对阿霉素肾病大鼠肾纤维化的保护作用及其可能机制。 方法 雄性SD大鼠48只,随机分为正常对照组、模型组、尿毒清组、贝那普利组,每组12只。采用左侧肾脏切除加尾静脉重复注射阿霉素的方法建立阿霉素肾病模型。术后1周尿毒清组给予尿毒清3 g&#8226;kg-1&#8226;d-1灌胃,贝那普利组予贝那普利4 mg&#8226;kg-1&#8226;d-1灌胃。用药4、8周后观察各组大鼠24 h尿蛋白量、血生化及光镜、电镜下肾脏病理变化,应用免疫组化方法测定肾组织骨调素（OPN）、纤连蛋白（FN）、胶原蛋白Ⅳ（COLⅣ）的表达。 结果 模型组大鼠24 h尿蛋白量、BUN、Scr、胆固醇（Cho）、三酰甘油（TG）水平及系膜肾小球系膜基质比（M/G）均显著高于正常对照组（P < 0.01）;血白蛋白（Alb）水平显著低于正常对照组（P < 0.01）;肾组织OPN、FN、COLIV的表达亦显著高于正常对照组（P < 0.01）。尿毒清组24 h尿蛋白量、BUN、Scr、Cho、TG水平及M/G均显著低于模型组（P < 0.01）;肾组织OPN、FN、COLIV表达亦显著低于模型组（P < 0.01）。 结论 尿毒清可减少蛋白尿的排泄,纠正脂代谢紊乱,改善肾功能,加强FN、COLIV的降解,抑制肾小球细胞外基质的过度积聚,从而减轻肾脏病理损害,发挥其肾脏保护作用。     

缬草油对高胆固醇血症大鼠肾间质纤维化的改善作用及机制探讨

目的观察缬草油对高胆固醇血症大鼠肾间质纤维化的改善作用并探讨其可能机制。方法将64只SD大鼠随机分为正常组、高脂组、缬草油组和辛伐他汀组,每组16只。缬草油组给予缬草油25mg·kg^-1·d^-1灌胃,辛伐他汀组同时给予辛伐他汀5mg·kg^-1·d^-1灌胃。比较各组大鼠总胆固醇（TC）、甘油三酯、低密度脂蛋白（LDL）、高密度脂蛋白、24h尿蛋白量、血肌酐（SCr）、肾间质损伤指数及转化生长因子β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）mRNA和蛋白表达。结果与高脂组相比,第16周时缬草油组肾小管间质损伤指数降低,TC、LDL、24h尿蛋白量和SCr降低,差异均有统计学意义（P〈0．01）。与正常组相比,第8周时,高脂组肾小管内TGF-β1表达增多,差异有统计学意义（P〈0．01）,而α-SMA表达未见明显升高;第16周时,TGF-β1表达更为明显,α—SMA表达明显增加,差异均有统计学意义（P〈0．01）。缬草油能显著降低各时间点TGF-β1、rSMA蛋白和mRNA表达,其改善作用较辛伐他汀更明显。结论缬草油可改善高脂血症大鼠肾间质纤维化,可能与其调节血脂,下调肾组织TGF-β1表达,抑制肾小管上皮细胞转分化有关。     

雷公藤内酯醇对IgA肾病大鼠蛋白尿和nephrin、podocin蛋白及mRNA表达的影响

目的:观察雷公藤内酯醇(TP)对IgA肾病(IgAN)大鼠尿蛋白和足细胞nephrin、podocin蛋白及mRNA表达的影响。方法:采用牛血清白蛋白+四氯化碳+脂多糖的方法建立IgAN大鼠模型。将60只大鼠随机分为正常组、模型组、洛丁新组、TP剂量组、TP中剂量组、TP高剂量组,每组10只。于0,10,14周收集血尿标本,并处死大鼠留取肾组织标本。检测24 h尿蛋白定量,血生化指标,分别采用实时定量PCR及ELISA法检测肾组织nephrin、podocin mRNA和蛋白表达。结果:(1)实验前各组大鼠尿蛋白差异无统计学意义(P>0.05),第10周末各造模组蛋白尿均明显高于正常组(P<0.01),第14周末TP各剂量组及洛丁新组蛋白尿均较模型组明显减少(P<0.01),TP高剂量组Alt值高于其余各组(P<0.05);(2)与正常组相比,模型组nephrin、podocin蛋白、mRNA表达明显降低(P<0.01);TP各剂量组及洛丁新组nephrin、podocin蛋白、mRNA表达均较模型组明显增高(P<0.05);TP中、高剂量组较洛丁新组nephrin、podocin蛋白浓度显著增高(P<0.05);TP各剂量组与洛丁新组及TP各剂量组间nephrin、podocin mRNA表达均差异无统计学意义(P>0.05)。结论:TP能够明显减轻IgAN大鼠的蛋白尿,且能明显提高肾组织nephrin、podocin mRNA及蛋白的表达。提示TP能调节IgAN足细胞相关分子nephrin、podocin基因及蛋白的表达,减少足细胞损害,减少尿蛋白。     

依贝沙坦对单侧输尿管梗阻小鼠肾脏整合素连接激酶表达的影响及意义

目的研究依贝沙坦（Irb）对单侧输尿管梗阻（UUO）小鼠肾脏整合素连接激酶（ILK）表达的影响,并探讨其与肾小管上皮间充质转化的关系。方法将雄性CD-1小鼠随机分为假手术对照组（C,n=20）、UUO组（UUO,n=40）和Irb治疗组（UUO＋Irb,n=40）,分别于术后1、3、7和14d处死小鼠。Masson染色观察肾间质纤维化。免疫组化检测小鼠肾组织ILK、上皮细胞钙黏蛋白（E-cadherin）和α平滑肌肌动蛋白（α-SMA）表达。Western印迹观察小鼠肾组织ILK蛋白表达。荧光实时定量PCR（real-time PCR）检测ILK、E-cadherin、α-SMA和纤连蛋白（FN）mRNA表达。结果与C组相比,UUO组术后1d ILK mRNA及蛋白表达均增高;术后3d FN mRNA表达上调,E-cadherin mRNA及蛋白表达明显减少,α-SMA mRNA及蛋白表达显著增加。与UUO组相同时间点比较,Irb治疗组ILK、FN及α-SMA表达均被显著抑制（P均＜0．05）;E-cadherin表达则增高（P＜0．01）。ILK蛋白表达与α-SMA蛋白表达呈正相关（r =0．707,P＜0．01）,与E-cadherin蛋白表达呈负相关（r=-0．919,P＜0．01）。结论Irb能减轻肾脏纤维化,抑制肾小管上皮细胞转分化,这可能与其抑制肾小管细胞ILK表达有关。     

肾脏局部醛固酮对糖尿病肾病大鼠肾小管间质转分化的作用

目的 探讨醛固酮对糖尿病肾病（DN）大鼠肾小管间质转分化的影响。 方法 采用Wistar大鼠腹腔注射链脲菌素（STZ,60 mg/kg）制备糖尿病模型,4周后尿蛋白>30 mg/d为DN模型成功（n=16）,随机分为DN组（n=8）和螺内酯组（SP组,n=8）,以另8只正常大鼠作为对照组（N组）。SP组给予螺内酯40 mg&#8226;kg-1&#8226;d-1,N组、DN组每日以等量清水灌胃。8周后处死大鼠,收集尿、血浆、肾组织检测24 h尿蛋白定量、血肌酐和肾脏病理变化;用放射免疫法检测血浆、肾组织醛固酮浓度;用免疫组化、Western印迹方法检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）蛋白的表达;用RT-PCR的方法检测E-cadherin、α-SMA mRNA的表达。 结果 与对照组比较,DN组尿蛋白排泄量、血肌酐均显著增加（均 P < 0.01）,肾组织E-cadherin蛋白和mRNA表达显著下调（均P < 0.01）,α-SMA蛋白和mRNA表达均显著上调（均P < 0.01）。DN组大鼠肾组织醛固酮显著升高[（24.71±5.30） ng/g比（16.38±2.85） ng/g,P < 0.01],与尿蛋白排泄量、血肌酐、α-SMA蛋白表达呈正相关（r = 0.737、0.574、0.688,均P < 0.05）,与E-cadherin蛋白表达呈负相关（r = －0.659,P < 0.01）。各组间血清醛固酮含量差异无统计学意义。与DN组比较,SP组大鼠尿蛋白排泄和血肌酐显著下降（均P < 0.01）,E-cadherin蛋白和mRNA表达上调（均P < 0.05）,而α-SMA蛋白和mRNA表达显著下调(均P < 0.01)。 结论 DN大鼠肾组织局部醛固酮参与了糖尿病肾病肾间质转分化,螺内酯可以阻断醛固酮与其受体结合,抑制肾小管间质转分化,从而起到肾脏保护作用。     

醛固酮-WNK4途径参与盐负荷对上皮钠通道的调节

目的 通过建立生理条件下的盐负荷饮食大鼠模型,观察醛固酮和WNK4在水盐代谢调节中的作用。 方法 将SD大鼠分为5组：高盐组（H,4% NaCl）、正常盐组（N,0.4% NaCl）、低盐组（L,0.07% NaCl）、高盐加醛固酮组（H+A,4% NaCl+1 mg&#8226;kg-1&#8226;d-1醛固酮）、低盐加螺内酯（L+S,0.07% NaCl+0.1 g&#8226;kg-1&#8226;d-1螺内酯）,所有大鼠自由饮水,喂养2周。用放射免疫法检测血浆醛固酮的变化。应用实时定量PCR和Western印迹法检测大鼠肾脏上皮钠通道γ亚基（γENaC）、WNK4的mRNA和蛋白的变化。 结果 H组大鼠血浆醛固酮水平低于N组（P < 0.05）,H+A组高于H组（P < 0.05）;L组大鼠血浆醛固酮水平高于N组（P < 0.05）,显示SD大鼠造模成功。L组大鼠肾脏γENaC蛋白表达高于N组,但是L+S低于L组;同时H组低于N组,H+A组高于H组,差异均有统计学意义（P < 0.05）。mRNA变化趋势和蛋白变化趋势一致。H组肾脏WNK4的蛋白表达高于N组,但是H+A组低于H组;同时L组低于N组,L+S组高于L组,差异均有统计学意义（P < 0.05）。mRNA的变化趋势和蛋白的变化趋势一致。 结论 饮食中的盐可以调节γENaC在肾脏的蛋白表达,醛固酮和WNK4都参与了机体对盐的调节,WNK4受到醛固酮的负调节作用。     

Expression and significance of Pdlim2 in the glomerular podocyte of hyperlipidemic rats

Objective To investigate the expression changes and significance of Pdlim2 in theglomerular podocyte of hyperlipidemic rats. Methods Forty-five individuals of SD rats were divided randomly into 3 groups (n＝15 in each group). The control group was fed with normal diet. The high fat group was fed with high fat diet. The simvastatin group was fed with high fat diet plus with simvastatin gavage (10 mg·kg-1·d-1). Five rats were sampled randomly from each group at week 4, 6, and 10 and the urinary protein excretion, the concentration of serum cholesterol, and the concentration of low density lipoprotein cholestorol were determined, the glomerular podocyte damage in rats was detected by electron microscope, the expression of Pdlim2 protein was determined by immunohistochemistry and by Western blotting. Results The levels of serum cholesterol and low density lipoprotein cholestorol increased significantly in high fat group and simvastatin group at week 4 compared to that in control group(P＜0.05), and the level in simvastatin group was significantly decreased compared with that in high fat group(P＜0.05). The urinary protein levels of high fat group and simvastatin group were significantly higher than that in control group at week 10, and the level in simvastatin group was significantly decreased in high fat group, there was significant difference in each group of comparison(P＜0.05). Podocyte injury was detected by electronic microscopy in high fat group at week 4, and the injury became more serious as the treatment time increased. Podocyte injury in the simvastatin group was significantly less than that in the high fat group and the control group at week 10. The positive staining of Pdlim2 was mainly in the glomeruli and the expression of Pdlim2 of the high fat group was lower than the simvastatin group, and both were lower than the control group at week 10. The expression of Pdlim2 protein of high fat group was lower than that in the control group since week 4(P＜0.05). The expression of Pdlim2 protein of high fat group was lower than that in the simvastatin group(P＜0.05), and both were lower than the control group at week 10(P＜0.05). ConclusionsHypedipidemia induces podocyte injury before urinary protein, which is suggested to be associated with the decrease of Pdlim2 protein. Simvastatin reduces podocyte foot processes of high fat induced fusion, which may be through protecting the expression of glomerular Pdlim2.     

Early mechanisms of renal injury in hypercholesterolemic or hypertriglyceridemic rats

Hyperlipidemia in conjunction with uninephrectomy leads to renal injury in rats. It is unknown whether this is due to mesangial cell or podocyte injury and whether the injuries induced by hypercholesterolemia and hypertriglyceridemia share a similar pathogenesis. Therefore, renal effects of hypercholesterolemia were studied in male rats with dietary hypercholesterolemia compared with rats on a regular diet. Renal effects of hypertriglyceridemia were studied in female Nagase analbuminemic rats (NAR). Hypertriglyceridemia was reduced in NAR by ovariectomy. Both models were studied after uninephrectomy or sham operation. Dietary hypercholesterolemia had little effect on plasma triglycerides, whereas ovariectomy in the NAR had no effect on plasma cholesterol. However, an increase in intermediate density lipoprotein cholesterol was common to both models. Dietary hypercholesterolemia and uninephrectomy separately induced a similar increase in proteinuria after 13 wk, which was additive when these interventions were combined. At this stage, only a minimal increase was present in glomerular alpha-smooth muscle actin staining, a marker of mesangial cell activation, or in mesangial matrix expansion. Moreover, platelet-derived growth factor-B chain, a marker of mesangial cell proliferation, was not increased. However, podocyte injury was prominent as evidenced by podocytic de novo expression of desmin and ultrastructural changes. Glomerular macrophage counts were increased by hypercholesterolemia but not by uninephrectomy, and were not related to the level of proteinuria. Hypertriglyceridemia and uninephrectomy in female NAR induced an increase in proteinuria after 24 wk, which was also associated with an increase in podocyte desmin expression without any mesangial activation and proliferation or matrix accumulation. Hypertriglyceridemia, proteinuria, and the increase in desmin staining were largely prevented by ovariectomy. Interstitial myofibroblast activation and tubulointerstitial injury accompanied proteinuria in both models. These findings indicate that both hypercholesterolemia and hypertriglyceridemia aggravate renal injury primarily via podocyte rather than via mesangial cell damage. Such podocyte injury is accompanied by tubulointerstitial cell activation and injury.     

Hypercholesterolemia in rats induces podocyte stress and decreases renal cortical nitric oxide synthesis via an angiotensin II type 1 receptor-sensitive mechanism

Podocyte stress precedes proteinuria in hypercholesterolemic rats. Molsidomine, a nitric oxide (NO) donor, prevented podocyte stress and proteinuria in long-term hypercholesterolemia, suggesting that podocyte stress was due to NO deficiency. Podocytes express the angiotensin II type 1 receptor, which influences their function. Because NO counteracts angiotensin II, it was hypothesized that in a setting of impaired renal NO availability, angiotensin II receptor inhibition could prevent podocyte stress. For determining the effect of NO deficiency on podocyte stress, one group of female rats were fed 2% cholesterol and another group the arginine analogue N-omega-nitro-L-arginine (L-NNA; 40 mg/kg food) for 2 wk. Another group of rats that were fed 2% cholesterol also received the NO donor molsidomine (120 mg/L water) for 2 wk before and during cholesterol feeding. For determining the influence of angiotensin II in the setting of decreased renal NO availability, rats that were treated with cholesterol or L-NNA received the angiotensin II type 1 antagonist losartan (200 mg/L water) for 2 wk before and during cholesterol or L-NNA administration. Desmin staining and electron microscopy were used to monitor podocyte activation. Glomerular caveolin was quantified by immunohistochemistry. Renal cortical NO synthesis, NO synthase isoforms, and caveolin-1 protein mass were also measured. Both short-term cholesterol and L-NNA induced podocyte stress as evidenced by enhanced desmin staining and electron-dense fused foot processes. Podocyte stress was prevented by molsidomine in short-term hypercholesterolemia. Furthermore, losartan prevented podocyte stress in rats that were treated with cholesterol or with L-NNA. Finally, hypercholesterolemia decreased renal cortical NO synthase activity and increased caveolin-1 protein mass and glomerular caveolin staining, and these changes were also prevented by losartan. It is suggested that podocyte stress in these models of early injury results from angiotensin II, unopposed by the action of endogenous NO. This underscores the strategic role of angiotensin II blockers in early kidney disease.     

Proteinuria is preceded by decreased nitric oxide synthesis and prevented by a NO donor in cholesterol-fed rats

BACKGROUND: Hypercholesterolemia decreases nitric oxide (NO) availability in the circulation and induces podocyte activation and renal injury in rats. It is unknown whether hypercholesterolemia decreases renal NO availability. To dissociate the injury-independent effect of hypercholesterolemia on renal NO availability from secondary effects of proteinuria, increasing concentrations of cholesterol were administered. To determine whether podocyte activation and renal injury were associated with NO deficiency, molsidomine, an exogenous NO donor, was administered to hypercholesterolemic rats. METHODS: Female rats were fed 0, 0.5, 1, or 2% cholesterol for 24 weeks. Rats fed 2% cholesterol were also studied for two weeks. In addition rats fed 0 or 1% cholesterol received 120 mg molsidomine/L drinking water. Renal NO availability was determined by measuring renal NO synthesis and superoxide activity. Podocyte activation was monitored by desmin staining. RESULTS: Hypercholesterolemia dose-dependently increased proteinuria. In the absence of proteinuria, hypercholesterolemia decreased renal NO synthesis (4.2 +/- 0.5 in 0.5% cholesterol vs. 6.8 +/- 0.6 pmol/min/mg protein in controls; P < 0.05). With the exception of neuronal nitric oxide synthase (nNOS), renal NOS protein mass remained unaffected. Renal superoxide activity was dose-dependently increased, thus further lowering renal NO availability. Podocyte injury was dose-dependently increased even in the absence of proteinuria (score, 40 +/- 4 in 0.5% cholesterol vs. 9 +/- 4 in controls; P < 0.05). After two weeks, hypercholesterolemia caused no proteinuria, but did cause some podocyte injury. Renal NOS activity was decreased, but glomerular endothelial NOS (eNOS) staining was unchanged. Molsidomine prevented proteinuria, podocyte activation, and all further renal injury. CONCLUSIONS: Hypercholesterolemia decreases renal NO synthesis, and induces podocyte activation before proteinuria appears. Renal superoxide activity is increased once rats are proteinuric, further lowering renal NO availability. All of these changes can be prevented by a NO donor.     

X线造影剂对高胆固醇血症大鼠的肾毒性及己酮可可碱的保护作用

目的 比较不同渗透浓度X线造影剂对高胆固醇血症大鼠的肾毒性，探讨己酮可可碱（PTX）对造影剂肾毒性是否有保护作用。 方法 48只健康雄性SD大鼠，随机分为正常饮食组8只（NN组）和高胆固醇饮食组40只（H组，4%胆固醇+1%胆酸钠）。8周末，高胆固醇饮食组随机分为5组，每组8只，分别为高胆固醇饮食组（HN组）、高胆固醇+低渗造影剂组（HL组）、高胆固醇+等渗造影剂组（HI组）、高胆固醇饮食+高渗造影剂组（HH组）、高胆固醇+高渗造影剂+PTX组（HHP组）。在注射造影剂后48 h测定各组大鼠的血清总胆固醇、三酰甘油、血清肌酐、内生肌酐清除率（Ccr）、钠钾排泄分数及血管紧张素Ⅱ（AngⅡ）的浓度；光镜下观察肾组织学改变；免疫组化法检测肾组织NF-κB的蛋白表达；原位细胞调亡（TUNEL）染色检测肾小管上皮细胞的凋亡。 结果 所有给予高胆固醇饮食的大鼠血清总胆固醇水平明显增高（P < 0.05）。HH组的血清肌酐、钠钾排泄分数及血浆Ang Ⅱ水平均分别明显高于HHP组、HL组及HI组（P < 0.05）；HH组Ccr水平[（0.11±0.02） ml·min-1·（100 g）-1]则明显低于HHP组[（0.43±0.03） ml·min-1·（100 g）-1]、HL组[（0.25±0.02） ml·min-1·（100 g）-1]和HI组[（0.27±0.03） ml·min-1·（100 g）-1]（P < 0.05）。组织病理显示，HH组大鼠肾小管上皮细胞发生明显的变性和坏死，肾小管上皮细胞凋亡率（89.60%±6.40%）明显高于其他各组[NN组（2.40％±0.77％）、HN组（5.60％±1.08％）、HHP组（8.91％±1.44％）、HL组（63.34％±11.97％）、HI组（61.50％±9.40％）]（P < 0.05）。肾组织NF-κB阳性细胞的平均灰度值明显低于其他各组（P < 0.05）；而HL组和HI组间上述指标比较差异无统计学意义 （P > 0.05）。 结论 高胆固醇血症环境下注射不同渗透浓度X线造影剂均可致造影剂肾损害。PTX对高胆固醇血症环境下所致的造影剂肾毒性有保护作用。     

辛伐他汀对糖尿病肾病大鼠肾小管间质CTGF、β-catenin表达的影响

目的:观察辛伐他汀对糖尿病肾病(DN)大鼠肾小管间质结缔组织生长因子(connective tissue growth factor,CTGF)、β-连环蛋白(β-catenin)表达的影响。方法:将30只Wistar雄性大鼠随机分为正常对照组(N)、DN模型组(DN)、DN模型+辛伐他汀治疗组(DS)三组。利用腹腔注射链脲佐菌素建立DN大鼠模型;模型建立后,第4周、8周、12周,记录大鼠体重、检测血糖、测量24 h尿蛋白定量;12周末处死大鼠,检测血肌酐(Scr)、尿素氮(BUN)、胆固醇(TC);取大鼠肾组织行HE染色,进行病理组织学观察;用免疫组化法检测肾小管间质中CTGF及β-catenin的表达;采用实时荧光定量PCR技术检测肾组织中CTGF及β-catenin基因mRNA的表达。结果:实验12周末,与DN模型组比较,DS组大鼠24 h尿蛋白定量、Scr、BUN显著降低(P<0.05);肾组织病理改变减轻,免疫组化和实时荧光定量PCR结果均显示,肾小管间质CTGF、β-catenin的表达明显下调(P<0.05)。结论:辛伐他汀可同时下调糖尿病肾病大鼠肾小管间质CTGF及β-catenin的表达,降低蛋白尿,保护肾脏功能。提示辛伐他汀可能通过调节CTGF和Wnt/β-catenin信号通路的表达,发挥其延缓肾小管间质纤维化的作用。     

Expressions of transient receptor potential cation channel 6 and integrin-linked kinase in glomerular of proteinuric kidney diseases

Objective To observe the expressions and distribution of transient receptor potential cation channel 6 (TRPC6) and integrin-linked kinase (ILK) in the glomeruli of renal biopsy tissue of patients with proteinuric kidney diseases, and to investigate the effect of TRPC6 over-expression on ILK in vitro. Methods The archival histological specimens of patients admitted to Tangdu hospital from 2012 to 2013, with 24-hour urinary protein over 1 g, were collected. The expressions and distribution of TRPC6 and ILK in the glomeruli of renal biopsy tissue were observed by immunohistochemistry. MPC5 podocytes were cultured in vitro and they were stimulated with 10-7 mol/L ADR for 12, 24 and 36 h. The pcDNA3.1(+)-TRPC6 plasmid and pcDNA3.1(+) were transfected into MPC5 podocytes by liposome 2000 reagent to establish the TRPC6 overexpression group and the negative control group respectively. Western blotting was used to detect the expressions of TRPC6 and ILK protein. Results There were 14 cases of membranous nephropathy, 13 cases of focal segmental glomerulosclerosis (FSGS), 15 cases of membranoproliferative glomerulonephritis, 12 cases of mesangial proliferative glomerulonephritis, 10 cases of hyperplastic sclerosis nephritis, 15 cases of IgA nephropathy, 13 cases of purpura nephritis, 15 cases of lupus nephritis, 13 cases of hypertensive renal injury, 14 cases of diabetic nephropathy and 9 cases of normal renal tissue included. In glomerulus, TRPC6 was expressed mainly in podocytes, and the expressions of TRPC6 in these renal tissues were higher than that in normal renal tissues (all P＜0.05), except for hypertensive nephropathy. ILK was expressed in podocytes and the mesangial areas. The expressions of ILK in FSGS, lupus nephritis and diabetic nephropathy were higher than that in normal kidney tissue (all P＜0.05), while the other renal tissues was high but showed no statistical difference with normal kidney tissue (all P＞0.05). The expressions of TRPC6 and ILK were positively correlated in renal tissues of FSGS and diabetic nephropathy (r=0.906, P＜0.001; r=0.783, P=0.001 respectively). The expressions of TRPC6 and ILK protein in 24 and 36 h stimulating with ADR were significantly higher than that in the control group (all P＜0.05). The expression of ILK in the TRPC6 overexpression group was significantly higher than that in the normal control group (P＜0.05). Conclusions The expressions of TRPC6 and ILK increase in the glomeruli of patients with kidney diseases with proteinuria being the main manifestation, especially in FSGS and diabetic nephropathy. The up-regulation of TRPC6 can increase the expression of ILK protein, which may be involved in podocyte injury.     

缬草油对高胆固醇血症大鼠肾小管上皮细胞巢蛋白表达的影响

目的：探讨缬草油对高胆固醇血症大鼠肾小管上皮细胞巢蛋白表达的影响。方法：大鼠随机分为正常组、高脂组、缬草油（25mg·kg-1.d-1）组和辛伐他汀（5mg·kg-1.d-1）组,用含4%胆固醇和1%胆酸钠的高脂饲料饲喂大鼠建立高脂模型。观察8、12、16周时各组大鼠血脂、尿蛋白、血肌酐和肾小管间质病理改变,免疫组化法检测肾小管上皮细胞巢蛋白和α-平滑肌肌动蛋白（α-SMA）表达。结果：随时间延长,高脂大鼠肾小管上皮细胞逐渐出现巢蛋白和α-SMA表达。巢蛋白表达与总胆固醇、低密度脂蛋白、24h尿蛋白、血肌酐正相关（r=0.963、0.830、0.944、0.706,P〈0.01）,与肾小管间质损伤指数正相关（r=0.974,P〈0.01）,与α-SMA呈正相关（r=0.804,P〈0.01）。缬草油能显著降低大鼠血总胆固醇、低密度脂蛋白、24h尿蛋白和血肌酐,下调肾小管上皮细胞巢蛋白和α-SMA表达（P〈0.01）。在巢蛋白表达减少同时,肾小管间质损伤和纤维化明显改善,其改善作用较辛伐他汀更明显（P〈0.05）。结论：缬草油可能通过降脂、下调肾小管上皮细胞巢蛋白表达减轻高胆固醇血症大鼠肾间质纤维化。     

血管紧张素Ⅱ输注诱导肾小球ILK表达改变和肾小球损伤

目的观察血管紧张素II（AngII）输注及替米沙坦治疗对大鼠肾小球整合素蛋白激酶（Integrin-linked kinase,ILK）表达的影响,探讨AngII和ILK在肾小球损伤中的作用。方法将18只雄性SD大鼠随机分为3组：A组为对照组,由生理盐水代替AngII;B组用AngII以400ng·kg^-1·min^-1持续输注14d;C组在B组基础上加用替米沙坦30mg·kg^-1·d^-1进行干预。每组6只。每周末测量尾动脉收缩压、24h尿蛋白定量,于14d处死动物。心脏采血,检测血肌酐（SCr）;留取肾组织,行PAS染色,光镜下观察肾组织病理学改变;免疫组织化学法、RT-PCR及Western印迹法检测ILK表达。结果①AngII输注后,大鼠血压逐渐升高,尿蛋白持续增加,肾小球系膜区增生加重,替米坦治疗可以明显降低血压和减少尿蛋白（P〈0．05）,减轻肾小球系膜区增生（P〈0．01）。②AngⅡ输注14d时,ILK表达显著增加,替米沙坦治疗可显著降低ILK表达（P〈0．05）。结论肾脏ILK表达升高可能是AngII引起肾脏损害的重要机制。